RNA interference refers to the process of sequence-specific post-transcriptional gene silencing mediated by double-stranded RNA (dsRNA). After the discovery of the phenomenon in plants in the early 1990s, specific and selective inhibition of gene expression in an extremely efficient manner in Caenorhabditis elegans using dsRNA was reported (Fire et al., 1998). The sequence of the first strand (sense RNA) coincided with that of the corresponding region of the target messenger RNA (mRNA). The second strand (antisense RNA) was complementary to the mRNA. The resulting dsRNA turned out to be several orders of magnitude more efficient than the corresponding single-stranded RNA molecules (in particular, antisense RNA).
The process of RNAi begins when the enzyme DICER encounters dsRNA and chops it into pieces called small-interfering RNAs or siRNAs. This protein belongs to the RNase III nuclease family. A complex of proteins gathers up these siRNAs and uses their code as a guide to search out and destroy any RNAs in the cell with a matching sequence, such as target mRNA (see Bosher & Labouesse, 2000, Nat Cell Biol, 2000, 2(2):E31; and Akashi et al., 2001, Antisense Nucleic Acid Drug Dev, 11(6):359).
In attempting to use RNAi for gene knockdown, it was recognized that mammalian cells have developed various protective mechanisms against viral infections that could impede the use of this approach. Indeed, the presence of extremely low levels of viral dsRNA triggers an interferon response, resulting in a global non-specific suppression of translation, which in turn triggers apoptosis (Williams, 1997, Biochem Soc Trans, 25(2):509; Gil & Esteban, 2000, Apoptosis, 5(2):107-14).
In 2000, dsRNA was reported to specifically inhibit three genes in the mouse oocyte and early embryo. Translational arrest, and thus a PKR response, was not observed as the embryos continued to develop (Wianny & Zernicka-Goetz, 2000, Nat Cell Biol, 2(2):70). Research at Ribopharma AG (Kulmbach, Germany) reported the functionality of RNAi in mammalian cells, using short (20-24 base pairs) dsRNAs to switch off genes in human cells without initiating the acute-phase response. Similar experiments carried out by other research groups confirmed these results (Elbashir et al., 2001, Genes Dev, 15(2):188; Caplen et al., 2001, Proc. Natl. Acad. Sci. USA, 98:9742). Tested in a variety of normal and cancer human and mouse cell lines, it was determined that short hairpin RNAs (shRNAs) can silence genes as efficiently as their siRNA counterparts (Paddison et al., 2002, Genes Dev, 16(8):948). Recently, another group of small RNAs (21-25 base pairs) was shown to mediate downregulation of gene expression. These RNAs, small temporally regulated RNAs (stRNAs), regulate timing of gene expression during development in Caenorhabditis elegans (for review see Banerjee & Slack, 2002 and Grosshans & Slack, 2002, J Cell Biol, 156(1):17).
Scientists have used RNAi in several systems, including Caenorhabditis elegans, Drosophila, trypanosomes, and other invertebrates. Several groups have recently presented the specific suppression of protein biosynthesis in different mammalian cell lines (specifically in HeLa cells) demonstrating that RNAi is a broadly applicable method for gene silencing in vitro. Based on these results, RNAi has rapidly become a well recognized tool for validating (identifying and assigning) gene functions. RNAi employing short dsRNA oligonucleotides will yield an understanding of the function of genes being only partially sequenced.
Recently, Krutzfeldt and colleagues have shown that a class of specially engineered compounds called ‘antagomirs’ can effectively silence the action of microRNAs (miRNAs), non-coding pieces of RNA that regulate gene expression (Krutzfeldt et al., 2005, Nature, 438(7068):685-9).
The preceding is a discussion of relevant art pertaining to RNAi. The discussion is provided only for understanding of the invention that follows, and is not an admission that any of the work described is prior art to the claimed invention.
Although potential applications of siRNA technology are vast in medical science, their administration and delivery to the desired site of action proves a significant complication, due mainly to ubiquitous expression of RNAses. In fact, in the state of the art, delivery is achieved mainly, by direct injection into the desired tissue, this form of administration not being appropriate for treatment of many different diseases. For example, the treatment of intestinal conditions affecting the whole intestine or a significant portion, via the injection of formulations containing siRNAs into the intestinal tissue would be highly impractical, due to the dimensions of the organ.
This invention relates to a method for intestinal delivery of siRNA, and provides a specific example of the same comprising treatment of Crohn's disease via administration of IL-12 specific siRNA.
Interleukin-12 (IL-12) is a heterodimeric 70 kDa glycoprotein (IL12-p70) consisting of a 40 kDa subunit (designated IL12-p40) and a 35 kDa subunit (designated IL12-p35) linked by disulfide bonds that are essential for the biological activity of IL-12.
IL-12 is a key cytokine that regulates cell-mediated immune responses and type 1 T helper (Th1) cell inflammatory reactions (Gately et al., 1998 Annu Rev Immunol. 16:495-521; Trinchieri, 1998, Adv Immunol. 70:83-243).
One particular IBD is Crohn's disease, a pathology characterized by an increased production of IL-12 by antigen-presenting cells in intestinal tissue and interferon-γ and tumor necrosis factor α (TNF-α) by intestinal lymphocytes and macrophages (Fais et al., 1994, J Interferon Res. 14(5):235-8; Fuss et al., 1996, J Immunol. 157(3):1261-70; Monteleone et al., 1997 Gastroenterology, 112(4):1169-78; Parronchi et al., 1997 Am J Pathol. 150(3):823-32; Plevy et al., 1997, J Immunol. 15; 159(12):6276-82).
Crohn's disease causes inflammation in the small intestine. The inflammation can cause pain and can make the intestines empty frequently, resulting in diarrhoea. The most common symptoms of Crohn's disease are abdominal pain and diarrhoea, although rectal bleeding, weight loss and fever may also occur. Bleeding may be serious and persistent, leading to anaemia. Children with Crohn's disease may suffer delayed development and stunted growth.
Most people are first treated with drugs containing mesalamine, a substance that helps control inflammation. Sulfasalazine is the most commonly used of these drugs. Patients who do not benefit from it or who cannot tolerate it may be put on other mesalamine-containing drugs, generally known as 5-ASA agents, such as Asacol, Dipentum or Pentasa. Possible side-effects of mesalamine preparations include nausea, vomiting, diarrhoea and headache. Some patients take corticosteroids to control inflammation. These drugs are the most effective for active Crohn's disease, but they can cause serious side effects, including greater susceptibility to infection. Drugs that suppress the immune system are also used to treat Crohn's disease. Most commonly prescribed are 6-mercaptopurine and a related drug, azathioprine. Immunosuppressive agents work by blocking the immune reaction that contributes to inflammation. These drugs may cause side effects like nausea, vomiting, and diarrhoea and may lower a person's resistance to infection. Surgery to remove part of the intestine can help Crohn's disease but cannot cure it. Due to the side effects and the lack of effectiveness of the current treatments for Crohn's disease, researchers continue to look for more effective treatments.
Inhibiting the action of IL-12 has been shown to suppress development and clinical progression of disease in a multitude of experimental models of autoimmunity and chronic inflammation (Caspi, 1998, Clin Immunol Immunopathol. 88(1):4-13). These models include experimental autoimmune encephalomyelitis (EAE), experimental autoimmune uveitis (EAU), collagen-induced arthritis (CIA), autoimmune nephritis, insulin-dependent diabetes mellitus (IDDM) and different models for IBD (Vandenbroeck et al., 2004, J Pharm Pharmacol. 56(2):145-60). In these models, the role of endogenous IL-12 has been addressed by using IL-12p40 knockout mice or by administering anti-IL-12 antibodies.
In particular, targeting IL-12 with antibodies is an effective treatment for the intestinal inflammation in animal models of Crohn's disease (Mannon et al., 2004, N Engl J Med. 351(20):2069-79). Thus, mice with trinitrobenzene sulfonate-induced colitis have a Th1-mediated gut inflammation characterized by greatly increased production of IL-12, interferon-γ and tumour necrosis factor α (TNF-α). In mice, administration of a monoclonal antibody against IL-12 can result in the resolution of established colitis and, if given at the time of induction of colitis, can prevent inflammation (Neurath et al., 1995, J Exp Med. 182(5):1281-90).
Anti-interleukin-12 can also prevent and treat the spontaneous colitis seen in models of Th1-mediated inflammation such as mice that over-express the human CD3ε gene and mice deficient in interleukin-10 (Davidson et al., 1998; Simpson et al., 1998).
Data from an early phase 2 study provide some evidence that treatment with a monoclonal antibody against IL-12 p40 may induce clinical response and remission in patients with active Crohn's disease (Mannon et al., 2004). This treatment is associated with decreases in Th1-mediated inflammatory cytokines at the site of disease.
Previous evidence obtained from animal models, as well as the clinical effects of anti-IL-12 in patients with Crohn's disease (Mannon et al., 2004, N Engl J Med. 351(20):2069-79), highlight the importance of IL-12 as a target for future treatments for Crohn's disease.
siRNA targeting of IL-12 expression has already been used to obtain modified dendritic cells (DC) that might be used in a variety of therapeutic in vitro, ex vivo and in vivo methods to modulate T cell activity, and thus have use in therapeutic approaches for the treatment of immune disorders in a mammalian subject (WO 03/104456; Hill et al., 2003, J Immunol. 171(2):691-6). siRNA targeting of IL-12 expression in mature DC has revealed a critical role for IL-12 in natural killer cell interferon γ (IFN-γ) secretion promoted by mature DC (Borg et al., 2004, Blood 104(10):3267-75). Further, IL-12 p35 inhibitors including siRNA have surprisingly demonstrated to block differentiation of preadipocytes to adipocytes and triglyceride accumulation in adipocytes (WO 03/104495).
siRNA targeting IL-12p40 delivered by means of liposome encapsulation to murine peritoneal cavity has been reported to modulate the local and systemic inflammatory response after endotoxin challenge (Flynn et al., 2004, J Inflamm 1(1):4).